New strategies which improve the efficacy of radiation therapy and/or protect surrounding normal tissue from damage can have a major impact on treatment outcome. Recent studies have reinvigorated the hypothesis that the outcome of radiation therapy is dependent upon the anti-tumor immune response and strongly support the testing of new combinations of ionizing radiation with immunotherapy. Heat shock protein (HSP) biology has been central to our research for many years. This work led to a completely new vaccine paradigm based upon the chaperone potential of large HSPs (first discovered by our group) and their ability to stimulate powerful anti-tumor immunity. Large HSPs, including hsp110 and the glucose regulated protein 170 (grp170) are related molecular chaperones that are highly efficient in binding to large substrate proteins. Our team has demonstrated that clinically relevant tumor antigens (e.g., gp100, HER- 2/neu, etc.) can be complexed to these large stress-response proteins by heat shock generating a highly effective antigen-specific, immune response. In a significant demonstration of lab bench-to-bedside research, our preclinical results led to a successful RAID application from the NCI for the development of a novel hsp110-based vaccine now undergoing Phase I evaluation in advanced melanoma patients. In new preliminary and published data, we have discovered that grp170 and pathogen-associated molecules (PAMPs) can act synergistically to stimulate the innate immune response and this is dependent on the chaperoning activity of grp170. We believe that this discovery can result in a novel, multipronged approach to cancer therapy. Therefore, we have engineered a chimeric chaperone, termed Flagrp170, by fusing a truncated grp170 chaperone with bacterial flagellin-derived PAMP which has an NF-kB-activating domain. Since preliminary data show a strong potential for Flagrp170 to provide a radioprotective effect, we hypothesize that this novel agent could both enhance therapeutic responsiveness to ionizing radiation (through enhanced immune responses of the vaccine) and provide critical protection of normal tissue. We will test our hypothesis in three specific aims. In Aim 1, we predict that this chaperoning competent chimeric macromolecule can preferentially deliver selected antigens to specialized antigen-presenting cells and concurrently stimulate the NF-kB signaling for their functional activation. In Aim 2, we will test whether vaccination with Flagrp170 complexed with tumor antigen will generate a more potent and durable immune response than unmodified grp170. In Aim 3, the potential of using Flagrp170 to improve efficacy of ionizing radiation and provide tissue protection against radio-toxicity will be investigated. Successful completion of proposed research will not only provide a better understanding of the multifunctional features of the chimeric chaperone, but will also establish a scientific rationale for design the new generation of HSP-based vaccines to be used alone or in conjunction with the standard-of-care therapies (e.g., radiotherapy) in future clinical trials.